Project 2 - Dorothee Herlyn and Rajasekharan Somasundaram Human BRAFV600E (mutated BRAF) is a tumor-specific antigen expressed by >60% of melanomas, but not by normal cells except for nevi. Based on its tumor-specific expression, BRAF^600E provides a potential immunotherapeutic target for melanoma. BRAFV600E is expressed intracellularly and not on the surface of tumor cells, and therefore this mutation is a target for T cells, but not antibodies. The major goal of the studies proposed in Project 2 is to develop a mouse model of therapeutic targeting mouse BRAFV600E, using BRAFV600E-specific vaccines and small molecule inhibitors. In addition, as a direct clinical correlate, T cell responses of melanoma patients to stimulation with the mutated epitope will be determined. In preliminary studies, a transgenic mouse melanoma model of active specific immunotherapy against BRAFV600E was developed. Furthermore, the lymphocytes of 4 of 9 HLA-A2-positive melanoma patients proliferated after stimulation with BRAFV600E peptides predicted to bind to HLA-A2. In the studies proposed in the mouse melanoma model, various BRAFV600E vaccines, such as peptides, recombinant baculovirus-derived protein in different adjuvants or fused to GM-CSF will be tested for their potential to induce cellular immunity (proliferative, cytotoxic and delayed-type hypersensitive T lymphocytes) in mice (Aim 1). Selected vaccines will then be evaluated for their capacity to inhibit growth of BRAFV600E-positive mouse melanoma cells in vivo. The mechanism underlying tumor growth inhibition will be determined, with emphasis on the role of CD8+ and CD4+ T cells, memory T cells and epitope spreading, possibly induced by the vaccines. Potential synergistic effects of BRAFV600E-specific vaccines and small molecule inhibitors will be evaluated (Aim 2). Finally (Aim 3), the hypothesis that BRAFV600E induces cellular immunity in melanoma patients' peripheral blood mononuclear cells will be tested by stimulating the cells with BRAFV600E peptides or protein and measuring proliferative and cytolytic T lymphocyte responses. These studies provide the basis for clinical trials with BRAFV600E vaccines and inhibitors in melanoma patients. This project interacts with projects 3 and 4 in which recombinant proteins and small molecule inhibitors of BRAFV600E will be developed, and with project 1 in which the inhibitors will be selected for tumor growthnhibiting properties in vitro and in vivo using human and mouse BRAFV60DE positive melanoma cells as targets.